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ARTICLE |
CORRESPONDENCE Claude Sportès: csportes{at}mail.nih.gov
 Top ABSTRACT RESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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c, common chain; rh, recombinant human; MFI, mean fluorescence intensity; RTE, recent thymic emigrant; TREC, TCR excision circle. R.E. Gress and C.L. Mackall contributed equally to this paper.
© 2008 Sportès et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jem.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
Cytokines that signal via the common
chain (c) represent promising therapeutics based on their potential to augment T cell expansion and increase the effectiveness of immune-based therapies (1–6). Within this family, IL-2 is a prototypic activating cytokine, secreted by and selectively signaling activated T cells, as well as up-regulating its own receptor (IL-2R). In contrast, IL-7 is a prototypic homeostatic cytokine, produced constitutively by nonlymphoid cells. Its receptor (IL-7R
) is expressed on resting T cells, and then rapidly down-regulated after T cell activation or IL-7 signaling (7). IL-7 is essential for T cell development in mice (8–10) and humans (11, 12), as well as for T cell homeostasis, because it is required to maintain naive CD4+ and CD8+ T cells in vivo (13, 14). IL-7 levels rise in serum and tissues after T cell depletion and fall upon recovery (14–16).
In preclinical studies, IL-7 therapy exerts marked effects on T cell immune reconstitution in mice (17) and primates (18–20). IL-7 augments effector and memory responses to vaccination in mice (21) with preferential enhancement of responses to weak subdominant antigens. In preclinical models, IL-7 therapy augments antitumor responses, leading to improved survival when combined with antitumor vaccines (21, 22). The capacity for supranormal levels of IL-7 to augment T cell cycling in response to antigens with low affinity for the TCR appears largely responsible for homeostatic peripheral expansion, which involves increased T cell proliferation to self-antigens during lymphopenia (13, 23, 24). Many studies have documented a central role for homeostatic peripheral expansion in the immunorestorative effect of IL-7 therapy (19, 20, 25), whereas augmentation of thymic output has also been recently suggested (18). In regard to CD4+CD25Hi regulatory T (T reg) cells, IL-2 plays a major role in their development, maintenance, and expansion, whereas T reg cells express low IL-7R levels (26, 27) and recent work suggests that IL-7 therapy can expand CD4+ T cells without expanding T reg cells (28).
Lymphopenia induced by cytotoxic chemotherapy or other insults can significantly diminish immune function (29). In adults, CD4+ T cell recovery after severe immune depletion requires the reemergence of a pool of naive T cells, which requires 18–24 mo and may occur only in individuals younger than 40–45 yr (30, 31). A therapeutic approach to accelerate, or simply promote in older individuals, the recovery of a widely diverse T cell repertoire may find a multitude of clinical applications. If rhIL-7 therapy in humans can augment immune responses to weak antigens, hasten reconstitution of naive T cell populations, and spare T reg cell expansion in particular but not restricted to older individuals, it could improve the effectiveness of immune-based therapies for cancer or chronic infection in various populations with iatrogenic (chemotherapy), pathological (HIV), or physiological (aging) immune insufficiency. This study sought to characterize the immunobiologic effects of rhIL-7 therapy in humans and, specifically, its potential for immune rejuvenation of T cell subpopulations.
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and CD3+/CD16+ T cells increased to a similar extent as β T cells, whereas NK cells increased minimally at the two highest dose levels. Circulating B cells decreased during rhIL-7 therapy (Fig. S1 A). Pharmacologic serum levels of IL-7 were achieved in a dose-dependent manner documented on both the first and last day of treatment (Fig. S1 C). The lower serum levels achieved on the last day most likely reflects the much higher lymphocyte mass on that day. 5 out of 15 evaluable subjects developed anti–rhIL-7 antibodies at day 28 or 35, but importantly, none developed neutralizing antibodies as measured in a bioassay using an IL-7–dependent cell line, and no subject developed lymphopenia after treatment.
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expression. Despite continued administration of rhIL-7 through day 14 resulting in sustained increases in circulating IL-7 levels (Fig. S1 C), T cell cycling substantially declined after day 7 coincident with IL-7R down-regulation (Fig. 1 D) that, as in mouse models (7), did not simply reflect receptor blockade or internalization because it was associated with diminished mRNA transcription (Fig. 1 E).
Like other c cytokines, IL-7 also supports T cell survival by up-regulating members of the antiapoptotic bcl-2 family (32, 33), and rhIL-7 therapy induced significant increases in T cell bcl-2 expression in this study (Fig. 1 F); as bcl-2 has been well established in animal studies to diminish programmed cell death, this rise in bcl-2 expression may have contributed mechanistically to the increase in T cell numbers. Whereas cell cycling rates were tightly linked to IL-7R expression, bcl-2 up-regulation was sustained throughout therapy. At higher dose levels, bcl-2 expression persisted above baseline for at least 1 wk after discontinuation of rhIL-7 therapy. This sustained increase in bcl-2 expression was likely to be physiologically significant and contributed to the sustained increases in lymphocyte number observed, as similar effects have been seen in animals when sustained bcl-2 expression is induced.
To assess functionality of rhIL-7–expanded T cells, proliferation in response to TCR signaling in vitro was measured using titrated doses of plate-bound anti-CD3 (Fig. 3 A).
rhIL-7 therapy substantially increased maximal T cell proliferation, which peaked on day 14, the last day of therapy. Proliferative responses to fully mitogenic doses of anti-CD3 were approximately threefold greater than at baseline and, at very low doses of anti-CD3 stimulation, rhIL-7–treated subjects showed increases in proliferation greater than one log higher than those observed in normal donors or the subjects themselves at baseline. Thus, rhIL-7 therapy leads to transient, widespread T cell cycling that is tightly regulated by IL-7R expression, as well as a more sustained increase in bcl-2 expression, resulting in dramatic increases in T cell numbers persisting for several weeks after cessation of therapy. These rhIL-7–expanded T cells retain robust proliferative capacity, even to low level of TCR triggering, a finding that is consistent with IL-7's known capacity to costimulate TCR signaling and to mediate augmented responses to weak antigens (21).
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c cytokine routinely administered therapeutically to humans, also increases CD4+ T cell numbers in vivo (34, 35). However, IL-2–induced expansion of CD4+ T cell populations in humans involve selective expansion of CD4+CD25Hi, FOXP3+ T reg cells (34–36), as predicted by the CD25 expression that is the hallmark of this subset. Low level IL-7R expression on T reg cells (26, 27) suggests that IL-7 therapy may not selectively expand this subset. To assess whether CD4+ expansion was associated with preferential T reg cell expansion, FOXP3 mRNA was quantified in sorted CD4+ T cells before and after rhIL-7 therapy (Fig. 3 B). FOXP3 mRNA was not increased in CD4+ cells collected during the period of CD4+ expansion, nor was any significant FOXP3 mRNA detected in CD8+ cells at baseline or after therapy. These mRNA data are corroborated by multicolor flow cytometry analyses performed in selected patients from cohort 4, specifically addressing rhIL-7–induced proliferation in FOXP3+ and FOXP3– subsets. In summary, after 1 wk of treatment with rhIL-7, there is a mean 4.3-fold increase of the percentage of CD4+/FoxP3+/Ki67+ cells, whereas the mean fold increases for CD4+/FoxP3–/Ki67+ cells and for CD8+/FoxP3–/Ki67+ cells are 53.4- and 63.9-fold, respectively. After 2 wk of treatment, the mean fold increases are 1.6-, 24.1-, and 35.7-fold, respectively, whereas at week 3, 1 wk after the end of treatment, they are 0.2-, 5.9-, and 9.6-fold. Representative two-dimensional plots and overlay histograms in one patient are shown in Fig. 3 C. Thus, in contrast to rhIL-2, rhIL-7 therapy in humans induces robust CD4+ T cell expansion without selective expansion of T reg cells, which is consistent with a recent study from a separate cohort of subjects in another clinical trial of rhIL-7 (28). Furthermore, multiple studies have also demonstrated that in vivo IL-2 administration in humans has minimal effects on CD8+ T cells numbers (37, 38). In contrast, we show that the in vivo effects of rhIL-7 on the expansion of CD8+ T cells are, at a minimum, comparable if not superior to the effects on CD4+ T cells.
rhIL-7 increases TCR repertoire diversity
Because rhIL-7 receptor expression is high on resting, naive populations, we postulated that the rhIL-7–induced increase in circulating T cells could preferentially expand naive cells, and therefore lead to an overall increase in TCR diversity. TCR diversity spectratype analysis was performed on sorted CD4+ and CD8+ populations before and after (day 21) rhIL-7 therapy in 6 subjects treated at either 30 or 60 µg/kg, and the pre- and post-therapy spectratype divergence from a normal donor standard was assessed (see Materials and methods). Indeed, 4 of the 6 subjects tested had a statistically significant increase in repertoire diversity by 1 wk after the end of treatment (day 21) compared with baseline in CD4+, CD8+, or both T cell populations (Fig. 4).
The two other subjects showed a strong trend in at least one population. Such changes are surprising given the short time period that had elapsed since beginning rhIL-7 therapy, as eliciting spectratype profile differences would be difficult both in the case of individuals with an initially very diverse repertoire (hence, with little possibility of improvement) and of those with a severely depleted repertoire (with marked divergence from a normal donor standard).
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expression is tightly regulated during postthymic T cell differentiation, such that RTEs (25, 39), naive, and early memory populations (40) express high IL-7R levels, whereas effectors and late memory/senescent populations express lower levels (41, 42). To determine whether the effects of rhIL-7 therapy on TCR repertoire diversity could be explained by varied effects among peripheral T cell subsets with differential IL-7R expression, we measured cycling rates, bcl-2 expression, peripheral blood frequencies, and absolute numbers of RTEs, naive, effector, and memory populations before, during, and after therapy. Fig. 5 illustrates representative changes in a single subject (treated at 60 µg/kg), Fig. 6 summarizes findings across subjects and dose levels; detailed kinetics for all subsets are included in Fig. S2 (available at http://www.jem.org/cgi/content/full/jem.20071681/DC1).
Using CD45RA with CD27 or CCR7 coexpression to define the naive subsets, rhIL-7 therapy preferentially expanded naive CD4+ and CD8+ T cells, leading to increased frequency (Fig. 5, A and C) and absolute numbers (Fig. 6, A and D). Similarly, CD4+ RTEs defined by coexpression of CD45RA and CD31 substantially increased both in frequency and number after rhIL-7 therapy. In addition, modest expansion of the memory and effector subsets were observed (Fig. 6 A), which appeared to preferentially involve CD4+ central memory cells (Fig. 6 D). Of all subsets, the naive CD8+ expanded the most and significantly more than the CD8+ memory and effector subsets (P = 0.008), and these effects were similar regardless of subject age (Fig. S1 B). This preferential effect on naive cells, including RTEs, was not caused by differences in maximal Ki67 expression because rhIL-7 similarly increased the cycling rate of nearly all subsets in a steep dose-dependent manner (Fig. 5 B and Fig. 6 B) by day 7 of therapy, followed by a gradual return to near baseline by day 21 (Fig. 5 B and Fig. S2). Interestingly, however, whereas memory and effector subsets showed a brisk decline in Ki67 frequency by day 14, there remained a distinct subset of Ki67+ cells at this time point in both CD4+ and CD8+ naive subsets (Fig. 5 B). rhIL-7 also induced a dose-dependent increase in bcl-2 mean fluorescence intensity (MFI) in all subsets except CD8+ effectors (Fig. 6 C). Thus, rhIL-7 therapy increased total body T cell mass with preferential expansion of the naive subsets, resulting in a T cell pool in this adult population that more closely resembles that seen earlier in life. This could potentially relate to fine differential kinetics of cell cycling, IL-7R expression, or bcl-2 up-regulation, although changes in these parameters of similar magnitude were observed in nonnaive subsets as well (Fig. S2). Alternatively, these results could suggest that, after similar levels of rhIL-7 signaling, naive T cells undergo a greater proliferative burst than memory or effector subsets and/or that some component of the increase in naive subsets reflects input of cells from an alternative source.
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in the face of continued drug administration. The rhIL-7–expanded T cells remained functional, retaining robust responsiveness to TCR triggering with even a suggestion of increased sensitivity to TCR ligation after a suboptimal stimulus, as seen in preclinical models. rhIL-7 preferentially expanded naive CD4+ and CD8+ subsets, including the most naive circulating RTEs (CD4+/CD45RA+/CD31+), consistent with the results from mouse studies (44). The effects on CD4+ and CD8+ memory cells and CD4+ effectors were intermediate, whereas CD8+ effectors underwent little expansion. Notably, rhIL-7–expanded central memory CD4+ T cells, a subset that may be critically important in avoiding clonal exhaustion in the context of chronic persistent viral or tumoral antigenic exposure (47, 48). It is also noteworthy that rhIL-7 preferentially expanded the two subsets of T cells capable of initiating lymph node germinal center formation, the naive and central memory subsets.
Despite the clear evidence presented in this study that rhIL-7 therapy substantially increases TCR diversity by an increase in the size of the naive T cell pool, the contribution of the thymus to these rapid changes remains unclear. Preclinical studies provide evidence both for and against rhIL-7–induced augmentation of thymopoiesis (18–20). In this study, we observed increases in absolute numbers of TRECs (albeit modest) and in numbers of peripheral blood phenotypic RTEs. However, we saw neither evidence for thymic enlargement nor significant correlation between age and rhIL-7 effects. Furthermore, our TREC analysis on sorted naive CD4+/CD31+/CD45RA+ cells and naive CD8+/CD27+/CD45RA+ cells shows a pronounced dilution effect of TRECs consistent with the intense proliferation in these subsets, thus arguing that the observed effect on repertoire diversity is in large part caused by decreased skewing by proliferation of RTEs. Together, these data suggest that the observed changes reflected rhIL-7–induced increases in RTE cycling, as well as trafficking of RTEs from peripheral lymphoid tissue into the bloodstream as seen in the mouse model (25, 44), whereas no direct effect on thymopoiesis can be demonstrated in the short time span of this study. Indeed, in clinical settings, thymic participation in immune reconstitution in adults is delayed for several months after the depleting event (30), raising the possibility that more direct thymopoietic effects of rhIL-7, if induced, could require more chronic rhIL-7 administration. However, even if the observed rhIL-7 effects are thymic independent and reflect naive cell expansion rather than increased thymic throughput per se, the net effect of rhIL-7 therapy remains a substantial, measured, increase in TCR repertoire diversity in adult subjects, thereby reshaping their T cell pool. During the aging process, the accumulation of senescent and more oligoclonal CD8+ cells with a terminal differentiation phenotype has been associated with dysfunctional immune responses (49). The effects of rhIL-7 on naive and central memory subsets could restore balance in T cell subsets during immunological aging and restore a T cell profile resembling that seen earlier in life. Such "antiaging" effects could prove beneficial for T cell–depleted hosts or older hosts, where the combined loss of total T cell number, naive cells, and the corollary loss of repertoire diversity appears to contribute to diminished overall immune responsiveness.
In summary, in this first clinical trial of rhIL-7 initiated in humans, and after the brief study of another National Cancer Institute–initiated trial (28), we present evidence that rhIL-7 therapy in humans induces dramatic, polyclonal, prolonged CD4+ and CD8+ T cell expansion in vivo, with preferential increases in T cells bearing diverse TCR repertoire specificities. The effects are mediated primarily through increased peripheral T cell cycling and augmented cell survival. The rhIL-7–expanded T cells retain significant functional capacity, and the CD4+ T cell expansion is not accompanied by a disproportional increase in T reg cells, such as that which occurs after rhIL-2 therapy. rhIL-7 appears to be an effective T cell growth factor with "immune rejuvenating" properties that suggest it would be effective in augmenting immune reactivity in hosts with impaired immunity caused by physiological (age), iatrogenic (chemotherapy/transplantation), or pathological (HIV) lymphodepletion. In immunologically normal, as well as deficient hosts, rhIL-7's capacity to augment responses to weak antigens and to increase T cell cycling without T reg cell expansion may be clinically exploitable in the context of immunotherapy regimens for cancer and/or chronic infection. Future clinical studies of rhIL-7 in the context of immune-based therapies are warranted.
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MATERIALS AND METHODS |
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TopABSTRACTRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Flow cytometry and cell sorting
PBMC analysis.
We used flow cytometry to assess the intracellular and cell surface expression of selected markers on PBMCs at the specified time points. We analyzed EDTA anticoagulated peripheral blood specimens on the same day without cryopreservation; the specimens were handled according to established clinical guidelines. We stained 3 ml of the samples for 4-color flow cytometry using a whole-blood lysis method; the samples were analyzed on a FACSCalibur (BD Biosciences) using CellQuest software (BD Biosciences). We used an additional 5 ml of blood to generate mononuclear cells via density gradient separation for determination of intracellular Ki-67 expression in T cells and T cell subsets. We identified T cells and T cell subsets using directly conjugated monoclonal antibodies: anti-CD3, -CD4, -CD8, -/β TCR, and -/ TCR; T cells were further characterized using anti-CD9, -CD11a, -CD71, -CD127, -CD184, and -Ki-67. We evaluated B cells with anti-CD20 and NK cells with a combination of anti-CD16 and -CD56, evaluated on CD3– lymphocytes. We used directly conjugated, mouse IgG1 to ascertain background staining. We identified lymphocytes based on a gate established by forward and side-angle scatter, and confirmed using anti-CD45/-CD14 (Leukogate; BD Biosciences). The absolute number for each population was generated using absolute peripheral blood lymphocyte count, determined by a Cell Dyne Sapphire (Abbott). We used peripheral blood from healthy adult controls to establish 95% confidence intervals. We obtained all monoclonal antibodies from BD Biosciences, with the exception of anti-CD4, -CD8, -CD127, and -/β TCR (Beckman Coulter), anti-/ TCR (Pierce-Endogen), and anti-CD11a (Dako).
CD4+ and CD8+ T cell subset analysis
We further analyzed cryopreserved PBMC to examine T cell subsets, markers of cell proliferation and apoptotic susceptibility within these subpopulations. We stained cells with CD8-Pacific Blue, CD3-APC-Cy5.5, CD45RA-PECy5 (all Invitrogen), CD27-APC (eBioscience), CD4-APC-Cy7, and CD31-FITC (all BD Biosciences); fixed; permeabilized (BD Biosciences); stained for intracellular markers with isotype control-PE, Ki-67-PE or Bcl-2-PE (all BD Biosciences); and analyzed on a LSR II flow cytometer (BD Biosciences) equipped with violet 405, argon 488, and HeNe 635 lasers. We acquired data using DIVA software (BD Biosciences) and analyzed using FlowJo (Tree Star, Inc.). Gating on populations of CD3+CD8+ and CD3+CD4+ T cells, expression of Ki67 or bcl-2 was determined on T cell subsets defined by the following antibody expression patterns: RTEs as CD4+/CD45RA+/CD31+, naive CD4 or CD8 T cells as CD27+ CD45RA+, memory as CD27+CD45RA–, and effector T cells as CD27– CD45RA+/–. Alternatively, cells were stained with CCR7-FITC (R&D Systems), and the remaining surface markers and analyzed for naive (CCR7+CD45RA+), central memory (CCR7+CD45RA–), effector memory (CCR7–CD45RA–), and effector RA (CCR7–CD45RA+), following the research of Sallusto et al. (50). We calculated the absolute cells number/milliliter expressing a particular phenotype by multiplying the frequency of the subset within CD4 or CD8 T cells by the absolute number of those cells as calculated during cytometric analysis of fresh whole blood.
IL-7RA, FOXP3 message determination
We used electronically sorted CD4+ and CD8+ populations for RNA extraction. We extracted RNA using Trizol (Invitrogen) according to the manufacturer's instructions, and then reverse transcribed 500 ng of RNA using the SuperScript III First-Strand Synthesis System (Invitrogen). We performed quantitative PCR using the Taqman ABI Prism 7000 Sequence Detection System (Applied Biosystems) on cDNA samples for FOXP3 and IL-7RA expression, and normalized them to the housekeeping gene ACTB. We purchased probes for detection of FOXP3, IL-7RA, ACTB, and the Taqman Universal PCR Master Mix (Applied Biosystems) and used them according to the provided protocol. We generated standard curves to quantify FOXP3, IL-7RA, and ACTB by cloning FOXP3, IL-7RA, and ACTB PCR products into plasmids using Platinum Taq and TOPO TA Cloning kit (Invitrogen) as per the manufacturer's instructions. PCR conditions were a 94°C 2-min hot start followed by 94°C for 30 s, 58°C for 30 s, 72°C for 45 s for 30 cycles, and culminating in a final 72°C 7-min extension step. Primer sequences were as follows: ACTB, 5'-GAGCACAGAGCCTCGCCTTTG-3' (sense), 5'-CGCCCACATAGGAATCCTTCT-3' (antisense); FOXP3, 5'-GCACCTTCCCAAATCCCAGT-3' (sense), 5'-GCAGGCAAGACAGTGGAAACC-3' (antisense); IL-7RA proprietary ("HS00233682" from ABI).
T cell proliferation in response to anti-CD3 signaling
We thawed PBMC cryopreserved at each time point, and then incubated it for 2 d in 200 µl/well of 10% human AB serum/AIM-V medium on 96-well flat-bottomed plates (Nunc) coated with the designed concentrations of OKT3 (Orthoclone). We measured cell proliferation by incorporation of [3H]thymidine (1 µCi/well; ICN) after a 16–18-h pulse. Cpm reflects proliferation after anti-CD3 minus cell proliferation in the medium. We ran normal donor controls with each assay.
TREC analysis
For TREC analysis, we sorted CD3+CD4+ and CD3+CD8+ T cells and, in two subjects, CD4+/CD31+ and CD8+/CD45RA+ T cells from cryopreserved PBMC on a FACSVantage (D Biosciences). Real-time quantitative-PCR was performed using the 5'-nuclease (Taqman) assay on cell lysates on an ABI7700 system (Applied Biosystems) according to the protocol of Douek et al. (43). Cells were lysed in 100 µg/ml proteinase K (Boehringer-Mannheim) in 10 mM TRIS, and heated for 1 h at 56°C, and then for 10 min at 95°C. PCR reactions contained Platinum Quantitative PCR SuperMix-UDG with ROX (Invitrogen) with 500 nM of each primer, and 150 nM probe. PCR conditions were 95°C for 5 min, 95°C for 30 s, and 60°C for 1 min for 40 cycles. The forward and reverse TREC primers were 5'-CACATCCCTTTCAACCATGCT-3' and 5'-GCCAGCTGCAGGGTTTAGTG-3', respectively, and the probe FAM-5'-ACACCTCTGGTTTTTGTAAAGGTGCCCACT-TAMRA (Integrated DNA Technologies, Inc.). A standard curve (102–7 TREC copies/well) was plotted using a plasmid containing the TREC gene sequence (provided by D.C. Douek, National Institutes of Health, Bethesda, MD). We normalized TREC values to cell content by concurrent measurement of the single-copy gene, RNase P, using the TaqMan RNase P Control Reagents kit (Applied Biosystems). Samples were analyzed in duplicate, and the results were averaged and normalized as TRECs/200,000 copies RNaseP, equaling TRECs/100,000 cells.
T cell repertoire diversity evaluation and statistical evaluation of TCR β-chain variable region (Vβ) spectratypes
We performed spectratype analyses separately on CD4+ and CD8+ T cell subsets from 6 subjects (3 each of cohorts 3 and 4). We sorted CD3+CD4+ and CD3+CD8+ T cells to 98% purity from cryopreserved PBMC on a FACS Vantage (BDIS). We used 105 cells of each to isolate RNA using Trizol (Invitrogen) in the presence of carrier tRNA and glycogen as carrier, and converted to cDNA using Oligo-dT as a primer for reverse transcription (First Strand cDNA kit; Roche). We amplified cDNA in 22 separate PCR reactions (Vβ families 1–24, excluding Vβ 10 and 19), each containing 1 specific human Vβ subfamily primer coupled with an unlabeled consensus Cβ region primer, followed by a 10-cycle run-off nested PCR reaction, using a FAM-labeled internal BC primer (Integrated DNA Technologies, Inc.). Labeled run-off reaction Cβ primer and Vβ family-specific (forward) primers were as specified in Currier et al. (51), except for Vβ 5, 13, 15, and 16. These remaining Vβ primers and the Cβ primer were as from Puisieux et al. (52). The initial and run-off PCR used 0.75 U cloned PFU DNA polymerase (Stratagene), and PCR settings were hot start with 2 min at 95°C followed by 30 cycles of 95°C for 30 s, 55°C for 45 s, and 72°C for 1 min, followed a final extension at 72°C for 10 min. Run-off reaction product was mixed at 1:9 ratio with loading buffer, consisting of Hi-Di Formamide (Applied Biosystems) containing size standards GeneScan-500 ROX, Red A (Applied Biosystems) mixed at a 1:20 ratio. Reactions were heated for 2 min at 95°C, and products were run on an ABI sequencer 3130XI (Applied Biosciences). Peaks were identified in the final spectratype histogram, and the area underneath was determined using GeneMapper v. 3.7 software (Applied Biosciences).
Using this same methodology, we also analyzed each Vβ family of 10–15 normal donor T cells (CD4+ and CD8+ combined), thereby defining "normal donor standards." For each donor's spectratype, we aligned the peaks by nucleotide base pair number, and used the area under each of 13 potential peak locations to calculate a total area under the spectratype curve, and then a proportional area for each peak. We then averaged the proportional areas to establish a normal donor standard spectratype. As expected, as we added more donors to the determination, the mean curves approached a more Gaussian-like distribution.
For each study subject, we then quantified the "divergence score" of each Vβ family from its counterpart Vβ normal donor standard as the sum of the absolute value of the divergence in proportional area at each peak. Therefore, divergence scores were established for each Vβ family, for each patient, separately for CD4+ and for CD8+ cells, and for both at baseline (pre) and day 21 (post). We then tested the hypothesis that, for a given CD4+ or CD8+ population in a given subject, the day 21 (post) divergence scores were significantly smaller than the baseline (pre) divergence scores in a nonparametric Wilcoxon analysis (2-tailed). A P value <0.05 indicates a statistically significant increase in repertoire diversity toward normality between the baseline and day 21 studies
Finally, for each single Vβ family, we calculated a "diversity shift" as the difference between "pre" and "post" divergence scores, and we calculated the median of these diversity shifts (median diversity shift) for the set of 22 Vβ family of each T cell population (CD4+ or CD8+), a positive value indicating a shift toward greater repertoire diversity.
Statistical analyses
Anti-CD3 proliferation.
We performed statistical analyses using GraphPad Prism version 4.0a for Macintosh (GraphPad Software). Significant differences when comparing two groups was determined by two-tailed unpaired nonparametric Mann-Whitney test. P values were considered significant if <0.05.
Correlation with age.
We evaluated the statistical significance of the correlation of age with the percent increase of various lymphocyte populations, as well as with TRECs, with a Spearman's correlation test with a significant 2-tailed P value of <0.05 (GraphPad Prism version 4.0a for Macintosh; GraphPad Software).
Spectratyping analysis.
See previous section.
Online supplemental material
Fig. S1 shows time courses of various lymphocyte populations following rhIL-7 therapy. Fig. S2 shows time course on percent change over baseline in circulating cells, Ki67 expression, and Bcl-2 up-regulation for all studied subsets. The online version of this article is available at http://www.jem.org/cgi/content/full/jem.20071681/DC1.
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Acknowledgments |
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This research was supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute (NCI), and Center for Cancer Research and was made possible through a formal collaboration (Cooperative Research and Development Agreement [CRADA] #01649) between the NCI and Cytheris Inc., the investigational new drug holder and manufacturer of rhIL-7, currently developing recombinant human IL-7 for therapeutic use. As per the CRADA between NCI and Cytheris Inc., the NCI investigators designed and conducted all the research experiments and analyses presented in the article independently from Cytheris Inc.
Three co-authors have financial interest in Cytheris capital: M. Morre is the founder and Chief Executive Officer and J. Engels and R. Buffet are employees of Cytheris. All other co-authors have explicitly denied any conflict of interest.
Submitted: 8 August 2007
Accepted: 21 May 2008
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| TABLE OF CONTENTS |
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; n = 3); 30 µg/kg/d (dashed line with 
; n = 5); and 60 µg/kg/d (solid line with 
; n = 4). Mean value for each cohort (± the SEM) are plotted at the indicated time points. (A) The absolute lymphocyte count from complete blood counts (left) and flow cytometry–based frequency were used to determine circulating absolute CD3+/CD4+ and CD3+/CD8+ counts, absolute numbers ± the SEM (bottom graphs), and percent change in absolute numbers over baseline (top graphs) of the respective subsets shown on day 1 (total lymphocytes only), 7, 14, 21, and 28 for all treated subjects, as well as day 55–90 for subjects treated with 30 or 60 µg/kg/d. Baseline values represent the mean of four separate analyses performed in each subject within 2 wk before initiation of rhIL-7 therapy. (B) Spleen size: bidimensional product by CT scan obtained at the time points shown; percent changes from the pretherapy scan are plotted. (C–F) Baseline (1 value obtained on day 0) and day 7, 14, 21, and 28 data points were generated for CD3+/CD4+ (left) and CD3+/CD8+ subsets (right). (C) Ki-67 shows the percentage of cells in the respective subsets expressing Ki-67 via flow cytometry at the time points shown. (D) IL-7R
, n = 16). rhIL-7–treated subjects showed increased proliferative responses to anti-CD3 on day 7 (dotted line with 
), day 14 (dashed line with 
), and day 21 (dashed line with •) compared with normal donors and on day 7 and 14 compared with baseline values (dashed and dotted line with filled 
; P < 0.05, two-tailed Mann-Whitney test). (B) FOXP3 mRNA copies per 104AXTB mRNA were quantified from sorted CD3+/CD4+ cells as detailed in the Materials and methods. No significant change in FOXP3 mRNA was detected between baseline values (white) and values obtained at the time of CD4+ expansion (day 14 in gray and day 21 in black; P = NS). Mean ± the SEM of data from subjects enrolled at each dose level are shown: 3 µg/kg (n = 2), 10 µg/kg (n = 3), 30 µg/kg (n = 4), and 60 µg/kg (n = 5). (C) Two-dimensional plots (top) and overlay histograms (bottom) of Ki67 expression in T reg cells and other CD4+ and CD8+ T cells in an individual subject. Percent Ki-67+ cells are indicated in each frame. Pretreatment, shaded histograms; day 7, black; day 14, dark gray; day 21, light gray.
